Pancreatic cancer (PCa) is the 4th deadliest cancer in the US, with median life expectancy of less than a year. The nucleoside analog Gemcitabine (Gem) is commonly used as frontline treatment, but only extends patient survival by several months over other single agent or combination therapies. The combination of Gem + nab- paclitaxel was recently shown to extend survival by ~2 months over Gem alone. The FOLFIRINOX regimen is a formulation of multiple drugs that has shown promise by doubling (~4-6 months) survival of PCa patients over Gem alone; however, its high toxicity may limit the patients that can tolerate this regimen. Understanding and targeting the molecular mechanism responsible for chemoresistance is a rational approach to improve treatment outcomes. Inhibiting such pathways should improve drug response, and thus extend patient survival. Importantly, chemosensitizers may reduce the effective dose of chemotherapy and thus limit drug toxicity. Cancer cells rely critically on survival pathways that allow them to tolerate a broa range of extrinsic and intrinsic stresses that would normally block cell growth. Drugs such as Gem, paclitaxel, and FOLFIRINOX, inhibit DNA replication and chromosome segregation, two processes that are already destabilized in cancer cells. The survival pathways that cancer cells have evolved to tolerate genome instability are also responsible for allowing them to survive drug treatment. These pathways include those in DNA repair and cell cycle checkpoints, as they protect the cancer genome from accumulating deleterious mutations or inducing cell death. Less understood is the role of the epigenome in promoting survival of cancer cells to intrinsic and extrinsic stress; however alterations of DNA methylation patterns can have profound effects on global gene expression that may allow the tumor cell to rapidly respond to cellular stresses. We recently reported that Thymine DNA Glycosylase (TDG), a T:G mismatch repair enzyme, is also a major enzyme for active cytosine demethylation. TDG therefore is directly involved in DNA repair and maintenance of the epigenome. Indeed knockdown of TDG in tumor cells leads to S phase arrest, and accumulation of 5- carboxylcytosine (an intermediate in cytosine demethylation), and TDG-null mouse embryo fibroblasts are hypersensitive to Gem. Targeting TDG should therefore block both DNA repair and epigenome pathways that cancer cells use to survive Gem. Towards this goal, we have identified a collection of FDA-approved compounds that potently inhibit TDG in vitro. We propose in this R21 application to validate TDG as a drug target in combination with Gem, by using patient-derived PCa cells and xenografts. This is an exciting R21 proposal because of its high translational potential by dually targeting DNA repair and epigenome pathways.